1. Field of the Invention
The present invention relates to a protein, prolidase gene, and recombinant DNA and a method for producing prolidase.
2. Background Art
Prolidase (proline dipeptidase, imidodipeptidase, EC3.4.13.9, hereinafter referred to as “prolidase”) is an enzyme which hydrolyzes dipeptides having a proline or hydroxyproline residue at its C terminus.
Enzymatic properties of many types of prolidase, from mammalian to archaebacteria, have been heretofore determined, and their genes have also been analyzed. For example, known prolidase of mammalian species are those derived from mouse liver [nonpatent literature 1] and human liver [nonpatent literature 2], and known prolidase of bacteria are those derived from E. coli [nonpatent literature 3] and Lactobacillus delbrueckii [nonpatent literature 4]. Known prolidase of archaebacteria include those derived from Pyrococcus furiosus [nonpatent literature 5]. Genes of these types of prolidase have also been reported.
Examples of prolidase, which is known for its enzyme only but its genes are not reported, include those derived from Xanthomonas maltophilia (patent literature 1).
In Japan, Aspergillus oryzae and Aspergillus sojae, i.e., aspergillus (yellow-green koji mold), have been used in the production of fermented foods such as soybean paste, soy sauce, and rice wine from a long time ago. Based on their long history of use, these microorganisms are particularly important from an industrial point of view because of high enzyme productivity and high safety reliability.
In the Aspergillus including yellow-green koji mold, the Genbank (the gene database) contains information concerning only the gene sequence of prolidase derived from Aspergillus nidulans (ACCESSION; AJ 296646). However, prolidase activities of purified enzymes and their gene products have not yet been reported.                [Patent literature 1]        JP Patent Publication No. 9-249        [Nonpatent literature 1]        Biochim. Biophys. Acta, 1308 (1), 15-16 (1996)        [Nonpatent literature 2]        J. Biol. Chem., 264 (8), 4476-4481 (1989)        [Nonpatent literature 3]        Nucleic Acids Res., 18 (21), 6439 (1990)        [Nonpatent literature 4]        Mol. Gen. Genet. 247, 494-500 (1995)        [Nonpatent literature 5]        J Bacteriol., 180(18):4781-9, (1998)        
When producing soy sauce, a starting material is degraded into a polypeptide by protease, which is generated by Aspergillus, the polypeptide is further degraded, by leucine aminopeptidase from the amino terminus and by acidic carboxypeptidase from the carboxyl terminus, into low molecular weight peptides composed mainly of amino acids and dipeptides.
The acidic carboxypeptidase of Aspergillus generated is less likely to degrade when the substrate is a tripeptide. It is much less likely to degrade when the substrate is a dipeptide. In particular, peptides having proline at its carboxyl terminus are less likely to be degraded and, thus, these peptides are likely to remain in soy sauce.
In contrast, leucine aminopeptidase is less likely to degrade peptides or acidic peptides having glycine at its amino terminus. Accordingly, these peptides are likely to remain in soy sauce. When proline is present in the peptides, the function of leucine aminopeptidase is suspended because of the presence of Xaa-Pro-peptide bonds in which an amino acid is bound to the proline on its imino group side. In unrefined soy sauce, it is believed that prolyl dipeptidyl peptidase specifically acts on the Xaa-Pro-peptide to release Xaa-Pro, and this allows leucine aminopeptidase to function, thereby releasing and generating amino acids. 13 types of neutral peptides and 13 types of acidic peptides have been reported as dipeptides in soy sauce, the structures thereof have been also deduced, and many Xaa-Pro including Gly-Pro are recognized based on the above-mentioned mechanisms.
Accordingly, degradation of these Xaa-Pro dipeptides can improve the protein degradation rate of soy sauce and an enzymatically-hydrolyzed flavor enhancer. Further, since proline is an amino acid having taste-enhancing properties, taste can be altered, and improvement in flavor can also be expected if Xaa such as Glu-Pro and Asp-Pro are also amino acids with taste enhancing properties.
Ser-Pro, Thr-Pro, Ala-Pro, and Gly-Pro are identified as Xaa-Pro which remains in soy sauce. All of these Xaa are amino acids which enhance sweetness and flavor, and thus improvements in sweetness and flavor of soy sauce can be expected. Accordingly, prolidase derived from microorganisms which relatively easily mass-produce enzymes is desired, and isolation from highly safe microorganisms such as Aspergillus is further strongly desired.